BglI NEB#R0143S

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	Using the dimerization of the 30 repeat fragment of ELP1 in the second round of PRe-RDL as an example, the designated ‘A’ fragment was obtained by digestion of 4 μg of ELP1-30 with 10 U AcuI and 40 U BglI for 3 hours at 37°C (see Figure 2A) in NEB Buffer 2 (New England Biolabs; Ipswich, MA). The ‘B’ fragment was obtained by digestion of 4 μg of ELP1-30 with 8 U BseRI and 40 U BglI for 3 hours at 37°C in NEB Buffer 2.

Protocol tips

Using the dimerization of the 30 repeat fragment of ELP1 in the second round of PRe-RDL as an example, the designated ‘A’ fragment was obtained by digestion of 4 μg of ELP1-30 with 10 U AcuI and 40 U BglI for 3 hours at 37°C (see Figure 2A) in NEB Buffer 2 (New England Biolabs; Ipswich, MA). The ‘B’ fragment was obtained by digestion of 4 μg of ELP1-30 with 8 U BseRI and 40 U BglI for 3 hours at 37°C in NEB Buffer 2.

Publication protocol

PRe-RDL was used to recursively double the ELP1 gene that encodes (GVGVP)30 and the ELP2 gene that encodes (GXGVP)20 where X = A and G in a 1:1 ratio, which were obtained after a single round of concatemerization. Using the dimerization of the 30 repeat fragment of ELP1 in the second round of PRe-RDL as an example, the designated ‘A’ fragment was obtained by digestion of 4 μg of ELP1-30 with 10 U AcuI and 40 U BglI for 3 hours at 37°C (see Figure 2A) in NEB Buffer 2 (New England Biolabs; Ipswich, MA). The ‘B’ fragment was obtained by digestion of 4 μg of ELP1-30 with 8 U BseRI and 40 U BglI for 3 hours at 37°C in NEB Buffer 2. Both DNA digests were run on a low melting point agarose gel. The ‘A’ digestion resulted in 3 bands: 1586 bp, 1821 bp, and a 2341 bp (1891 + ELP) fragment. The 2341 bp band was excised from the gel and purified with Qiagen’s gel purification kit. The ‘B’ digestion resulted in 2 bands: 1891 bp, and a 3857 (3407 + ELP) fragment, and the 3857 bp band was excised from the gel, purified, and eluted in 30 μL of distilled, deionized water. Equimolar amounts of the A and B fragments at a total DNA concentration of 5 ng/μL in a volume of 20 μL were ligated by incubation with T4 DNA ligase at 20 °C for 1 h. Top10™ chemically competent cells were transformed with the ligation product, as described previously. E. coli transformants were screened by colony PCR and by diagnostic restriction digests on an agarose gel. Each sequence was then confirmed by DNA sequencing.

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